Article Figures & Data
Figures
- FIG 1
VSV-EBOV vaccination elicits transcriptional changes suggestive of antiviral innate immunity and B-cell activation. (A) The bar graph depicts the number of differentially expressed genes (DEGs; defined as those with a fold change [FC] of ≥2 and a false-discovery rate [FDR]-corrected P value of ≤0.05) that have human homologues detected in animals vaccinated 3, 7, 14, and 21 days before challenge relative to animals vaccinated 28 days before challenge on the day of challenge (DPC 0). Red indicates upregulated DEGs, while blue indicates downregulated DEGs. The Venn diagram displays overlap between DEGs detected in day −3- and day −7-vaccinated animals. (B) Functional enrichment of DEGs detected in the day −3- and day −7-vaccinated animals. Color intensity represents the statistical significance (shown as –log10 of the FDR-corrected P value); the range of colors is based on the lowest and highest –log10 (FDR) values for the entire set of Gene Ontology (GO) processes. The number of DEGs enriching to each GO term is listed within each box; blank boxes represent lack of significant enrichment to a given GO term. (C) Heat map representing expression (shown as normalized RPKM values) of DEGs detected in the day −3- and day −7-vaccinated animals on the day of challenge. The range of colors is based on scaled and centered RPKM values of the entire set of genes: red represents increased expression, while blue represents decreased expression. Each column represents 1 animal; BL represents the transcriptional profile of the day −28 animals at 0 DPC. (D) Network showing direct interactions between DEGs detected on the day of challenge in the day −7 group enriched to the GO term “Immune system process.”
- FIG 2
Animals vaccinated 3 days before challenge display distinct transcriptional responses that correlate with disease outcome. Shown is a heat map representing hierarchical clustering of genes significantly associated with clinical outcome identified by maSigPro on 0 and 3 DPC. Clusters of genes that are coregulated are numbered 1 to 10. The range of colors is based on rlog transformation of normalized read counts: red represents increased expression, while blue represents decreased expression. Each column represents the nonviremic survivor (S), viremic survivor (VS), or viremic nonsurvivor (VNS).
- FIG 3
EBOV-Makona challenge 1 week after vaccination induces a recall response. (A) Number of DEGs detected in day −7-vaccinated animals following EBOV-Makona challenge. A Venn diagram displays overlap between DEGs detected 14 and 28 DPC in the day −7 group. (B) Functional enrichment of upregulated DEGs detected 14 and 28 DPC in the day −7 group. (C) Heat map representing upregulated DEGs detected 14 and 28 DPC in the day −7 group that enriched to “T-cell differentiation,” “B-cell activation,” and “Adaptive immune response.” Each column represents 1 animal; BL represents the transcriptional profile of the day −28 animals at 0 DPC.
- FIG 4
Animals that were vaccinated at least 2 weeks prior to EBOV challenge show limited gene expression changes. (A) Number of DEGs detected following EBOV-Makona challenge in animals vaccinated 28, 21, and 14 days before challenge. (B to D) Functional enrichment of DEGs observed in animals vaccinated 28 (B), 21 (C), and 14 (D) days before challenge. (E) Heat map of DEGs detected 28 and 42 DPC in the day −28 group that enriched to “Cell cycle” or “Response to stress,” respectively. Each column represents 1 animal. (F) Heat map of DEGs detected 42 DPC in the day −21 group that enriched to “Response to stress.” Each column represents 1 animal. (G) Heat map representing DEGs detected 28 DPC in the day −14 group that enriched to the GO term “Cellular macromolecule metabolic process.” Each column represents 1 animal.
Supplemental Material
FIG S1
Study timeline and ImmGen and DCQ analysis of transcriptional changes detected 7 days post-VSV-EBOV vaccination. (A) Study time line. (B) Heat map showing expression profile of DEGs detected 7 DPV in the day −7 group across various immune cell populations as predicted by ImmGen’s MyGeneSet application. Red indicates high while blue indicates low likelihood of expression within indicated immune cell population. B, B cells; T, T cells; NK, natural killer cells; DC, dendritic cells; MF, macrophages; MO, monocytes. (C) Changes in immune cell subsets based on DEGs detected in the day −7 group on the day of challenge were predicted using Immquant software, which implements the DCQ deconvolution algorithm using the IRIS database of gene expression. * and ** denote P values of ≤0.05 and ≤0.01, respectively, compared to baseline (BL) by unpaired t test. Mean and SEM are shown. Download FIG S1, TIF file, 1.4 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S2
Surviving animals in the day −3 group exhibit transcriptional changes consistent with resolution of immune response. (A) Number of DEGs in the 2 animals from the day −3 group that survived challenge 14, 28, and 42 DPC. (B) Functional enrichment of DEGs detected 14 and 28 DPC in the surviving animals from the day −3 group. Color intensity represents statistical significance (shown as −log10 of the FDR-corrected P value). The range of colors is based on the lowest and highest –log10 (FDR) values for the entire set of GO processes, the number of DEGs enriching to each GO process each day is listed within each box, and blank boxes represent lack of significant enrichment to a given GO term (C) Heat map of DEGs detected 14 DPC in the surviving animals from the day −3 group that enriched to “Immune system process” with a fold change (FC) of >6. Each column represents 1 animal; BL represents transcriptional profile of the day −28 animals at 0 DPC. (D) Heat map of DEGs detected 28 DPC in the surviving animals from the day −3 group that enriched to “Immune system process” with an FC of >3.8. Each column represents 1 animal; BL represents the transcriptional profile of the day −28 animals at 0 DPC. Download FIG S2, TIF file, 1.5 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S3
Animals vaccinated 1 week before challenge exhibit large transcriptional changes associated with adaptive immunity. (A) Functional enrichment of DEGs detected 3 and 6 DPC in day −7-vaccinated animals. (B) Heat map of common DEGs detected 3 and 6 DPC in the day −7 group that enrich to “Immune system process.” Each column represents 1 animal; BL represents day −28 animals at 0 DPC. (C) Immquant DCQ analysis using the IRIS database of gene expression was used to predict changes in immune cell subsets based on DEGs detected 14 DPC in the day −7 group. *, **, ***, and **** denote P values of ≤0.05, ≤0.01, ≤0.001, and ≤0.0001, respectively, at 14 DPC compared to baseline (BL) by unpaired t test. The mean and SEM are shown. (D) Functional enrichment of downregulated DEGs detected 14 and 28 DPC in the day −7 group. (E) Heat map of the most downregulated DEGs detected 14 and 28 DPC in the day −7 group that enriched to “Myeloid leukocyte activation” with an FC of >5. Each column represents 1 animal; BL represents the transcriptional profile of the day −28 animals at 0 DPC. Download FIG S3, TIF file, 1.4 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S4
Negative-control animals exhibit gene expression changes consistent with EVD. (A) Number of DEGs following EBOV-Makona challenge in animals that received VSV-MARV. The line graph represents the number of normalized viral transcripts (RPKM). *, P ≤ 0.05 compared to DPC 0. A Venn diagram displays overlap between DEGs detected 3 and 5 or 6 DPC in negative-control animals. (B) Functional enrichment of upregulated DEGs detected 3 and 5 or 6 DPC. (C) Network image of DEGs upregulated 5 or 6 DPC in negative-control animals that enriched to the GO term “Immune response,” with an FC of ≥5, and which directly interact with each other. Download FIG S4, TIF file, 2.0 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S5
Negative-control animals display gene expression changes consistent with lymphopenia and exhibit distinct transcriptional changes compared to naïve EBOV-Makona-challenged animals. (A) Functional enrichment of downregulated DEGs 5 or 6 DPC. Horizontal bar graphs represent the number of genes that mapped to each GO term listed, while the line graph represents the FDR-corrected P value. (B) Heat map of genes downregulated 5 or 6 DPC in negative-control animals that map to “TCR signaling” and “Cell cycle” (fold change of ≥4). Each column represents 1 animal. (C) Functional enrichment of DEGs detected 6 DPC in animals challenged with EBOV-Makona (19) or animals given VSV-MARV prior to EBOV-Makona challenge (present study). Color intensity represents the statistical significance; bubble size is based on the number of DEGs enriched. Download FIG S5, TIF file, 1.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
